Gamma-amino butyric acid type A (GABAA) receptors mediate the majority of fast synaptic inhibition in the brain. GABAA receptors are heteropentameric ligand gated ion channels. The majority of GABAA receptors are composed of 11, 22 and 32 subunits arranged in an 12123 counter clockwise fashion around a central ion conducting pore. In response to agonist binding this intrinsic channel opens, allowing chloride ions to flow through the membrane in a process called activation. The channel can close in response to agonist unbinding (deactivation) or in the continued presence of agonist (desensitization). Alterations in these macroscopic kinetic processes are seen in seizure disorders and in response to clinically important drugs such as benzodiazepines and barbiturates. Preliminary data suggests single residue mutations in the 1 subunit of the GABAA receptor can alter the rise time of activation and the extent of desensitization. Time constants of macroscopic activation, deactivation and desensitization, as well as the microscopic channel opening and closing rates of these mutant receptors, will be measured by patch clamp recordings. Utilizing in silico kinetic modeling and experimental data, the microscopic rate constants underlying these kinetic abnormalities can be deduced. Furthermore, the effects of mutations in each of the two 1 subunit can be separated by the use of linked, concatemeric subunits that can confine mutations to a single alpha subunit. Data from mutations isolated to the 1-2 interface or the 1-3 interface are of importance as the two subunit interfaces have different ligand binding properties. Knowing the specific effects on microscopic kinetics conferred by these mutations and any differences between mutations at individual 1 subunits will allow us to develop hypotheses about conformational movements abrogated by mutations in these regions. These experiments may advance our mechanistic knowledge of disease states and pharmaceutical modulators of the GABAA receptor. PUBLIC HEALTH RELEVANCE: Chemical signaling in the brain is controlled by ion channels such as the GABAA receptor. GABAA receptors have been implicated in anxiety, insomnia, seizure disorders and Alzheimer's disease and are the targets of pharmaceuticals aiming to treat these diseases. The proposed research will increase our understanding of the timing of chemical signaling via the GABAA receptor and will advance our knowledge of the role of GABAA receptors in these disease states and their treatments.